The Research On The Relevance Between The Changes Of Sodium Pump Activity And Endoplasmic Reticulum Stress In The Mechanism Of Reperfusion Injury In Isolated Rat Hearts

Objective: To explore the relationship between the changes of sodium pump activity and endoplasmic reticulum stress(ERS)in the mechanism of ischemia/reperfusion(I/R)injury in isolated rat hearts.Methods: The male SD rats were randomized into 6 groups(n=10):normal control group(NC),I/R group(I/R),ouabain-I/R group(OUA-I/R),anti-digoxin antiserum-I/R group(Anti-Dig-I/R),PP2(Src kinase inhibitor)-ouabain-I/R group(PP2-OUA-I/R),U73122(PLC inhibitor)-ouabain-I/R group(U73122-OUA-I/R);The isolated rat hearts were perfused on the Langendorff apparatus.Except for NC group,all the hearts were subjected to 30 min global ischemia and followed by 120 min reperfusion.The cardiac function indexes were recorded by biological signal collection and analysis system;Collected the coronary effluent at the same time and measured the coronary flow(CF)and the levels of lactate dehydrogenase(LDH),creatine kinase(CK)in the coronary effluent;The incidence of arrhythmias and arrhythmias score were determined;The myocardial infarct size was measured by TTC staining,The pathologic morphology was observed by HE staining;The activity of Na~+-K~+-ATPase in myocardial tissue was measured by ELISA;The content of mitochondrial calcium was determined by colorimetry;The flow cytometry was used to detect the level of intracellular free calcium fluorescence intensity and the apoptosis rate;The expressions of sodium pump ?1 subunit,endoplasmic reticulum molecular chaperon GRP78,ERS-associated apoptosis proteins CHOP and BCL-2/BAX were determined by Western blot.Results: 1 Effects of the changes of sodium pump activity on the recovery rate of cardiac functionDuring reperfusion: Compared with NC group,the recovery rate of LVSP*HR,±dp/dtmax were obviously reduced in I/R group(P 0.01);Compared with I/R group,the recovery rate of LVSP*HR,±dp/dtmax were decreased significantly in OUA-I/R,PP2-OUA-I/R and U73122-OUA-I/R group,while these were significant higer in Anti-Dig-I/R group(P 0.01);Compared with OUA-I/R group,both PP2 and U73122 partly abolished the effect of ouabain to reduce the recovery of cardiac function(P 0.01).2 The arrhythmia score in each groupDuring the balance perfusion in each group were not any arrhythmia;During reperfusion for 5min to 15min: Compared with NC group,the arrhythmia score was obviously increased in I/R group(P<0.01);Compared with I/R group,the arrhythmia score was increased significantly in OUA-I/R,PP2-OUA-I/R and U73122-OUA-I/R group,while arrhythmia score was significant lower in Anti-Dig-I/R group(P<0.01);Compared with OUA-I/R group,both PP2 and U73122 partly inhibited the effect of ouabain to worsen the pre-existed cardiac arrhythmia(P<0.01).3 Effects of the changes of sodium pump activity on the CFDuring reperfusion: Compared with NC group,the CF was obviously reduced in I/R group(P<0.01);Compared with I/R group,the CF was decreased significantly in OUA-I/R,PP2-OUA-I/R and U73122-OUA-I/R group,while CF was significant higher in Anti-Dig-I/R group(P<0.01);Compared with OUA-I/R group,both PP2 and U73122 partly inhibited the effect of ouabain to reduce the CF(P<0.01).4 Changes of LDH and CK activity in coronary effluentDuring reperfusion: Compared with NC group,the activity of CK and LDH was obviously increased in I/R group(P<0.01);Compared with I/R group,the coronary flow was increased significantly in OUA-I/R,PP2-OUA-I/R and U73122-OUA-I/R group,while the activity of CK and LDH was significant lower in Anti-Dig-I/R group(P<0.01);Compared with OUA-I/R group,both PP2 and U73122 partly inhibited the effect of ouabain to increase the content of CK and LDH(P<0.01).5 Determination of the degree of myocardial infarctionCompared with NC group,myocardial infarction of I/R group was significantly increased(P<0.01);Compared with I/R group,myocardial infarction was increased significantly in OUA-I/R,PP2-OUA-I/R and U73122-OUA-I/R group,myocardial infarction of Anti-Dig-I/R group was significant reduced(P<0.01);Compared with OUAI/R group,both PP2 and U73122 partly inhibited the effect of ouabain to increase the myocardial infarction size(P<0.01).6 Pathological morphology of myocardial tissueHE staining showed that myocardial fibers of rats in the NC group was neat and myocardia cell morphology was normal without edema and necrosis;Swelling and patchy necrosis of the myocardial cells,myofibrillar fragmentation were found in I/R group;Edema and necrosis of cells and myofibril fragmentation observed in OUA-I/R group were much more serious than that of control group,while the situation of rats in Anti-Dig-I/R group significantly improved;Compared with OUA-I/R group,both PP2 and U73122 partly inhibited the effect of ouabain to aggravate the degrees of cardiac tissue injure.7 Comparison the activity of Na~+-K~+-ATPase in myocardial tissueCompared with NC group,the activity of Na~+-K~+-ATPase in I/R group was significantly decreased(P<0.01);Compared with I/R group,the activity of Na~+-K~+-ATPase was reduced significantly in OUA-I/R,PP2-OUA-I/R and U73122-OUA-I/R group,while Na~+-K~+-ATPase activity of Anti-Dig-I/R group was significant increased(P<0.01);Compared with OUA-I/R group,both PP2 and U73122 partly blocked the effect of ouabain to restrain the activity of Na~+-K~+-ATPase(P<0.01).8 Changes of Ca2+ concentration in mitochondria and the lever of intracellular free calcium fluorescence intensityCompared with NC group,the level of mitochondrial calcium and calcium fluorescence intensity increased obviously(P<0.01);Compared with I/R group,the content of calcium and fluorescence intensity was increased significantly in OUA-I/R,PP2-OUA-I/R and U73122-OUA-I/R group,while the situation of Anti-Dig-I/R group was reduced significantly(P<0.01);Compared with OUA-I/R group,both PP2 and U73122 partly inhibited the effect of ouabain to elevate the calcium content(P<0.01).9 Changes of myocardial apoptosis index in each group.Compared with NC group,there was significantly increased in cardiomyocytes apoptosis in I/R group(P<0.01);Compared with I/R group,the apoptosis index was significantly increased in OUA-I/R,PP2-OUA-I/R and U73122-OUA-I/R group;While the apoptosis index was lower in Anti-Dig-I/R group(P<0.01);Compared with OUA-I/R group,both PP2 and U73122 partly interrupted the effect of ouabain to increase the apoptosis index(P<0.01).10 Changes of sodium pump ?1 subunit,GRP78,CHOP and BCL-2/BAX protein expressions in each groupCompared with NC group,the expressions of sodium pump ?1 subunit and BCL-2 were significantly decreased and GRP78,CHOP,BAX expressions were higher in I/R group(P<0.01);Compared with I/R group,the expressions of sodium pump ?1 subunit and BCL-2 were significantly decreased and GRP78,CHOP,BAX expressions were higher in OUA-I/R,PP2-OUA-I/R and U73122-OUA-I/R group;While sodium pump ?1 subunit and BCL-2 expressions were higher and GRP78,CHOP,BAX expressions were lower in Anti-Dig-I/R group(P<0.01);Compared with OUA-I/R group,both PP2 and U73122 partly blocked the effect of ouabain to down-regulate the expressions of sodium pump ?1 subunit and BCL-2 and up-regulate the expressions of GRP78,CHOP and BAX(P<0.01).Conclusion: Sodium pump function changes and ERS all participated in the process of myocardial I/R injury in isolated rat hearts.These findings suggested that sodium pump mediating ERS by activating signaling Src and PLC pathway may be one of the mechanisms of apoptosis induced by I/R injury.